The invention concerns the preparation of purified nucleic acid and its use especially in gene therapy.
Replicatable nucleic acid is usually produced by amplifying replicatable plasmid DNA in gram-negative bacteria such as e.g. E. coli. After lysis of the biomass (usually alkaline lysis with lysozyme or ultrasound), it is centrifuged and the supernatant is shaken out with phenol. Subsequently an ultracentrifugation on a caesium chloride gradient is carried out (Birnboim and Doly, Nucleic Acid Res. 7 (1979) 1513-1523, Garger et al., Biochem. Biophys. Res. Comm. 117 (1983) 835-842). However, such preparations contain endotoxins, phenol, caesium chloride and/or ethidium bromide as a dye.
A further process is described in the QIAGEN(copyright) Plasmid Handbook (Qiagen Inc., Chatsworth, USA) and EP-B 0 268 946. According to this process the cell lysate obtained after a conventional lysis is chromatographed on QIAGEN(copyright)-TIP, which contains QIAGEN(copyright) resin (a support material based on silica). The disadvantage of this process is that DNA binding proteins are not completely detached from the DNA and therefore the purified plasmid fraction contains proteins and in particular endotoxins (from the membrane of the gram-negative host cells) in considerable amounts. In another process after alkaline lysis of the E. coli biomass the centrifugation supernatant is chromatographed according to Birnboim and Doly under high salt conditions over anion exchange columns (e.g. Mono-Q, Source-Q from Pharmacia, Macroprep-Q from BioRad, Poros-Q from Perseptive Biosystems or HyperD-Q from Biosepra, cf. Chandra et.al., Analyt. Biochem. 203 (1992) 169-172; Dion et al., J. Chrom. 535 (1990) 127-147). Also in this case the purified plasmid fraction contains proteins and in particular endotoxins in considerable amounts.
In another process after alkaline lysis and subsequent phenol/chloroform extraction it is possible to chromatograph by gel filtration (McClung and Gonzales, Analyt. Biochem. 177 (1989) 378-382; Raymond et al., Analyt. Biochem. 173 (1988) 125-133). Even after this purification the plasmid preparation contains impurities and in particular phenol.
A process for the isolation and purification of nucleic acids for use in gene therapy is described in WO 95/21177 in which the purification is essentially carried out by centrifugation, filtration, affinity chromatography or chromatography on an inorganic chromatographic material with subsequent chromatography on an ion exchanger. An additional removal of endotoxins can then be achieved according to WO 95/21177 when the nucleic acid is treated with an endotoxin removal buffer which contains 10% Triton(copyright)X100 and 40 mmol/1 MOPS buffer (3-morpholino-1propanesulfonate buffer). A disadvantage of this process is that the nucleic acid purified in this manner contains impurities of Triton(copyright) and MOPS buffer. Although endotoxins can be removed by this process to a content of ca. 100 EU/mg DNA (Qiagen News 1/96, 3-5), it is not possible to remove endotoxins to a greater extent by this process.
However, for a therapeutic application such as for example for gene therapy a nucleic acid preparation is required which is as free as possible of all impurities (in particular substantially free of endotoxins). Above all the endotoxin content of plasmid preparations has been hitherto an unsolved problem as described for example by Cotten et al., Gene Therapy 1 (1994) 239-246. A reduced endotoxin content (ca. 100 EU/mg DNA) can only be achieved by the state of the art such as for example according to WO 95/21177 if the nucleic acids are treated with non-ionic detergents such as e.g. Triton (endotoxin removal buffer from WO 95/21177). However, Triton(copyright) has a biological action such as e.g. lung changes or reduction of blood pressure (Fiedler, xe2x80x9cLexikon der Hilfstoffe fxc3xcr Pharmazie und Kosmetik und angrenzende Gebiete (Band 9, 3rd edition, 1989, Editio Cantor, Del.)). The MOPS buffer which is additionally required also contains a substance that is problematic with regard to a therapeutic application.
The invention provides a nucleic acid preparation, preferably a plasmid DNA, of high purity in which endotoxins are substantially removed and preferably without ethidium bromide, phenol, caesium chloride, polymyxin or non-ionic detergents and also provides a simple and effective process for purifying such nucleic acids in particular for removing endotoxins.
The invention concerns a nucleic acid that can be replicated in gram-negative bacteria, preferably a plasmid DNA with a content of less than 1% protein, preferably less than 0.1% protein and a content of less than 1 EU/mg DNA, preferably 0.01-0.1 EU/mg DNA of endotoxins. This plasmid DNA is preferably free of ethidium bromide, phenol and caesium chloride, free of detergents based on octylphenolpoly(ethylene glycol ether)n such as Triton(copyright) detergents and also free of MOPS buffer substance and RNAse.
Amplification is understood as an increase in the copy number of a nucleic acid (in particular DNA and plasmid DNA) based on the replication of a vector. In this process numerous copies are produced from a template. A vector is replicated which represents the nucleic acid or which contains the cloned nucleic acid.
A plasmid DNA is understood as an extrachromosomal DNA duplex molecule. The size of a DNA plasmid is usually 1 to more than 200 kb and one to several hundred copies are present in host cells. Plasmid DNA is usually amplified in gram-negative bacteria such as e.g. E.coli and subsequently isolated. Plasmids are often used to construct cloning vectors, for the transfection of prokaryotic and eukaryotic cells. A therapeutic use is of especial importance inconnection with in vivo and ex vivo gene therapy. Plasmid DNA that is used therapeutically preferably has a length of 5 to 20 kb, particularly preferably 5-10 kb and is double-stranded. The plasmid DNA can be linearized or circularly closed. Preferably DNA is used that is essentially circularly closed.
Consequently the invention additionally concerns a pharmaceutical composition containing a nucleic acid according to the invention, preferably plasmid DNA, in a therapeutically effective amount and optionally additional pharmaceutical auxiliary substances, fillers or additives.
Endotoxins are lipopolysaccharides from gram-negative bacteria. Endotoxins can have a pyrogenic effect in mammals and induce an endotoxin shock. The main toxic component of endotoxins is lipid A, the polysaccharide moiety mediating the water solubility and the lipid moiety having the toxic effect. The biological effect of endotoxins in mammals are in particular a hypersensitization as well as other reactions which are accompanied by fever.
Plasmid DNA is amplified by standard methods in E. coli i.e. a gram-negative bacterium. After fermentation the biomass obtained in this process is lysed and the cells are lysed. In this process the endotoxins are released from the cell membrane. This means that after amplification of nucleic acids, in particular of plasmid DNA, in gram-negative bacteria and in particular in E. coli it is necessary to remove endotoxins if it is intended to use this plasmid DNA therapeutically.
Depending on the application doses of 50 xcexcg to 10 mg and more are used or planned for a therapeutic application of replicatable nucleic acids, in particular of plasmid DNA. The dose amount depends on the disease and type of administration. In an aerosol, e.g. for the treatment of cystic fibrosis, doses of 400 xcexcg and more are used. This applies likewise to plasmid DNA encapsulated in a lipid complex (e.g. in liposomes). In order to provide such amounts of replicatable nucleic acid that can be used therapeutically, it is necessary to produce the replicatable nucleic acid on a large scale. For this fermentation preparations are expedient with 1-5 kg biomass from which 1-5 g nucleic acid can be isolated.
The invention also concerns a process for the production of a plasmid DNA with a content of less than 1 EU/mg DNA, preferably 0.01-0.1 EU/mg DNA of endotoxins which is characterized in that plasmid DNA is replicated in gram-negative bacteria, the biomass is lysed and the soluble components are chromatographed on hydroxylapatite and subsequently the said plasmid DNA is isolated. Before chromatography on hydroxylapatite it is preferably to carry out an ion exchange chromatography which essentially removes RNA and foreign proteins. This can optionally remove further impurities and achieve a content of nucleic acid of less than 1% protein, preferably less than 0.1% protein, free of ethidium bromide, phenol and caesium chloride. Such a preparation is also preferably free of detergents based on octylphenol poly(ethylene glycol ether)n and MOPS buffer substance.
The process according to the invention enables numerous impurities to be avoided or removed which plasmid DNA contains if it is produced by a process familiar to a person skilled in the art. Above all it is surprisingly possible to drastically reduce the endotoxin content in a simple manner.
In the process according to the invention an outstanding removal of endotoxins is achieved by the chromatography with hydroxylapatite. This is all the more surprising since chromatography on hydroxylapatite is only used in the literature to separate DNA and RNA (Johnson and Ilan, Analyt. Biochem. 132 (1983) 20-25).
The chromatographic effect of hydroxylapatite is essentially based on the interaction between calcium2+ groups and the negative charge of the nucleic acid to be purified and to a lesser extent on the interaction of the nucleic acid to be purified with PO43xe2x88x92 groups on the surface of crystalline hydroxylapatite (cf e.g. Protein Purification Methods, Ed. by Elv. Harries and S. Angal, Oxford University Press 1989, 238-244). Chromatography on hydroxylapatite can be essentially referred to as an ion exchanger step for nucleic acids in which the bound DNA cannot be eluted from the hydroxylapatite matrix by a simple increase of ionic strength (e.g. NaCl) but rather by increasing the concentration, preferably of phosphate or citrate, divalent metal ions and/or EDTA.
In the process according to the invention endotoxins and the nucleic acid to be purified are firstly bound to hydroxylapatite primarily via dipole-dipole interactions in the chromatography on hydroxylapatite (e.g. HA-ceramic, BioGel HPHT, Bio-Gel HT/HTP from Biorad DE, HA-Ultrogel from IBF or HA spheroidal from BDH, Macrosorb C from Sterling Organics). The equilibration is usually. carried out at neutral pH in phosphate buffer. A denaturing agent is preferably added, as in the subsequent washing of the column. Surprisingly it is possible to displace the nucleic acid from its binding to hydroxylapatite with phosphate, citrate or calcium ions whereas the endotoxins remain bound. Instead of calcium, the displacement of the nucleic acid from its binding to hydroxylapatite can be achieved with other divalent metal ions which can replace calcium in the apatite such as e.g. Mg, Fe, Mn. For the elution the ion concentration is preferably 100 mmol/l or more. Ion concentrations between 100 and 500 mmol/l or 200 and 500 mmol/l are particularly preferred. A solution containing phosphate ions (e.g. phosphate buffer) is particularly preferably used. Before the elution (without denaturing agent) it is expedient to wash (with denaturing agent). It has turned out to be advantageous to for example use a phosphate of sulfate solution (100-200 mmol/l) to which a denaturing agent (e.g. urea or guanidine hydrochloride) has been added at a DNA-denaturing concentration (e.g. 6 mol/l urea).
In a preferred embodiment an ultrafiltration is additionally carried out after the chromatography on hydroxylapatite.
In order to produce the nucleic acid according to the invention the plasmids which represent or contain the nucleic acid are usually amplified in gram-negative bacterial cultures. Such methods are known to a person skilled in the art and are described for example by Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press and by F. M. Ausubel et al., eds. (1991), Current Protocols in Molecular Biology, Wiley Interscience, New York. For this the bacterial cultures which contain the plasmids are firstly subcultured and subsequently cultured in a suitable medium optionally with addition of a selection agent.
The biomass is also lysed by methods familiar to a person skilled in the art (mechanical or enzymatic lysis, see e.g. Birnboim and Doly, Nucleic Acids Research 7 (1979) 1513-1423) without addition of RNAse. It is possible to omit shaking out with phenol if the proteins are separated by chromatography on an anion exchanger. After lysis and separation of the insoluble components, preferably by centrifugation and filtration over a filter candle (5 xcexcm pores), the cell supernatant is preferably firstly chromatographed on an anion exchanger to remove proteins. Suitable anion exchangers are anion exchangers based on agarose such as for example Q-Sepharose. Other suitable anion exchangers are based on polymethacrylate (Macroprep/Bio-Rad, Germany), polystyrene/divinylbenzene (Poros/Perseptive, HyperD/Biosepra, Source/Pharmacia) or silica gel on the surface of which diethylaminoethyl (DEAE) or dimethylaminoethyl (DMAE) groups are for example bound.
In order to optimize the purification effect, the nucleic acid is eluted by means of a high salt gradient e.g. NaCl gradient (preferably 0.65 mol/l-0.85 mol/l) in TE buffer. This surprisingly enables numerous impurities (RNA, protein) to be separated in one step.
It is also preferable to carry out an additional isopropanol/ethanol precipitation, preferably after the hydroxylapatite chromatography, to minimize the bioburden and for desalting. Subsequently the nucleic acid according to the invention can be bottled under sterile conditions.